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Description

This kit is an Enzyme-Linked Immunosorbent Assay (ELISA) coupling with biotin-Streptavidin system, for quantitative measurement of dsRNA with length above 60 base pairs(bp), regardless of the sequence. The plate has been pre-coated with anti-dsRNA antibody. dsRNA present in the sample is added and binds to antibodies coated on the wells. And then biotinylated anti-dsRNA antibody is added and binds to dsRNA in the sample. 

After washing, HRP-Streptavidin is added and binds to the Biotinylated anti-dsRNA antibody. After incubation unbound HRP-Streptavidin is washed away. Then TMB substrate solution is added and catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the target amount of dsRNA captured in plate. The absorbance is measured at 450 nm.  

Specifications 

This kit is for quantitative measurement of residual dsRNA.

Kit Components

Transportation and storage

1.For unused kit: The whole kit could be stored at 2~8℃ in shelf life. Strong light should be avoided for storage stability.

2.For used kit: Once the microplate is opened, please cover unused wells with plate sealer and return to the foil pouch containing the desiccant pack, zip-seal the foil pouch and return to 2~8℃ as soon as possible after use. 

3.Other reagents should be returned to 2~8℃ as soon as possible after use. 

Materials Required 

• Microplate reader with 450±10nm filter (better if can detect at 450 and 650 nm wavelength).
• 37℃ incubator.
• RNase-free tips and centrifuge ( You will also find these materials at Gentaur.)

protocol

1. Determine the number of strips required for the assay. Insert the strips in the frames for use. Remaining plate strips not used in this assay should be repacked in the bag with desiccant. Close the bag tightly for refrigerated storage.
2. Add 100μL each of dilutions of standard, blank and samples into the appropriate wells. Cover with the plate sealer. Incubate for 1hr at room temperature with shaking at 500rpm.The samples should be diluted with STE buffer to appropriate concentration for accurate assay.
3. Wash step: Aspirate the solution and wash with 250μL wash buffer to each well and let it stand for 30s. Discard wash buffer completely by snapping the plate onto absorbent paper. Totally wash 4 times.
4. Add 100μL of biotinylated detection antibody working solution into each well. Cover with the plate sealer. Incubate for 1hr at room temperature with shaking at 500rpm.
5. Repeat wash step.
6. Add 100μL of HRP-streptavidin working solution into each well. Cover with the plate sealer. Incubate for 1hr at room temperature with shaking at 500rpm.
7. Repeat wash step again.
8. Add 100μL of TMB substrate solution into each well. Cover with the plate sealer. Incubate for 30 min at R.T.  Protect from The liquid will turn blue by the addition of substrate solution.
9. Add 50μL of stop solution into each well. The liquid will turn yellow by the addition of stop solution. Then run the microplate reader and conduct measurement at 450nm immediately. 

Performance

1. Sensitivity:  lower limit of detection： 0.001pg/μL

                           lower limit of quantitation：0312 pg/μL(for UTP-, pUTP-, N1-Me-pUTP-dsRNA), 0.125 pg/μL(for 5-OMe-UTP-dsRNA).

1. Precision：CV of Intra-Assay≤10%，CV of Inter-Assay≤10%
2. Recovery：80%~120%
3. Linearity：0312-1pg/μL(for UTP-, pUTP-, N1-Me-pUTP-dsRNA), 0.125-4 pg/μL(for 5-OMe-UTP-dsRNA)